Total analyte quantitation

ABSTRACT

Methods for determining total analyte concentrations and amounts, especially in combination with analyte separations are provided. Microfluidic devices are used to separate analyte mixtures and detect the individual analytes. Signal areas are summed for each individual analyte to quantitate the total analyte amount. Separate measurements of the total analyte sample are also used to determine total analyte concentration.

CROSS-REFERENCES TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. §§ 119 and/or 120, and any other applicablestatute or rule, this application claims the benefit of and priority toboth U.S. Ser. No. 60/198,511, filed on Apr. 18, 2000 and U.S. Ser. No.60/224,975, filed on Aug. 16, 2000, the disclosures of which areincorporated herein by reference.

COPYRIGHT NOTIFICATION

Pursuant to 37 C.F.R. § 1.71(e), Applicants note that a portion of thisdisclosure contains material which is subject to copyright protection.The copyright owner has no objection to the facsimile reproduction byanyone of the patent document or patent disclosure, as it appears in thePatent and Trademark Office patent file or records, but otherwisereserves all copyright rights whatsoever.

BACKGROUND OF THE INVENTION

Biological molecules, such as proteins and nucleic acids are routinelyfractionated and characterized, e.g., by capillary electrophoresis orusing microfluidic separation technology. For example, U.S. Pat. No.5,948,227 by Dubrow, entitled “Methods and Systems for PerformingElectrophoretic Molecular Separations,” describes methods forelectrophoretically separating molecular and macromolecular species inmicrofluidic devices.

Electrophoretic forces are typically used to separate materials inmicrofluidic devices, e.g., relying upon the electrophoretic mobility ofcharged species within an electric field applied to the material.Electrophoretic movement is used to separate mixtures of components asthey move through a microfluidic channel. Signal peak area of separatedcomponents is typically used to assess the extent of reactions, reactionrate constants, concentration of reactants, products, separatedcomponents, and a variety of other chemical and biochemical parameters.

Just as in traditional capillary electrophoresis, electrokinetic sampleintroduction in a microfluidic device biases sample introduction. Theelectric fields can cause preferential movement of reagents due todifferences in their mass to charge ratio, e.g., highly chargedmaterials move to the front or back of a fluid plug. This effect isdesirable when attempting to electrokinetically separate variouscompounds, but inhibits the ability to obtain measurements relating toentire samples, e.g., unseparated unbiased samples. For example, it isoften desirable to identify the total concentration, e.g., of nucleicacids or proteins, in a sample in addition to the concentration of eachnucleic acid fragment, e.g., after separation.

The calculation of kinetic constants in flowing systems has also beendescribed. For example, published PCT application WO 98/56956, byKopf-Sill et al., entitled “Apparatus and Methods for CorrectingVariable Velocity in Microfluidic Systems,” describes methods ofdetermining concentration, e.g., after an electrokinetically biasedsample introduction, using variable velocities, e.g., of reactants andproducts. This reference also describes, e.g., the use of gatedinjections to achieve representative sample aliquots and other relatedphenomena.

The present invention provides methods and apparatus for obtainingrepresentative or unbiased sample aliquots that are used to determine,e.g., total analyte concentrations. The methods and apparatus of thepresent invention provide these features and many others that will beapparent upon complete review of the following disclosure.

SUMMARY OF THE INVENTION

The present application provides methods for separating two or moreanalytes, e.g., in a microchannel, and determining a total analyteconcentration for the two or more analytes. For example, a sample, e.g.,a mixture of nucleic acid fragments, is optionally separated, theconcentration or amount of each individual analyte in the sampledetermined, and the concentration of all analytes together determined.The methods typically involve summation of individual analyteconcentrations using a representative sample aliquot, or alternatively,measurement of the total analyte concentration prior to separation.

In one aspect, a method for separating analytes and determining a totalanalyte concentration or amount is provided. The method comprisesflowing at least two analytes through a first channel, e.g.,electrokinetically or under pressure. The analytes typically flow intoan intersection of the first channel with a separation channel. After aspecified time, the analytes at the intersection are injected into theseparation channel. The analytes are then separated, e.g.,electrophoretically, resulting in two or more separated analytes. Theanalytes are then detected, resulting in, e.g., two or more signals. Thetwo or more signals are used to determine the total analyteconcentration, e.g., by summation of the two or more signals, e.g.,summation of the signal peak areas or peak heights corresponding toindividual analyte concentrations. The concentration or amount of eachindividual analyte is also optionally determined, e.g., to produce aratio of the amount of at least one of the analytes to the total analyteamount or to a portion of the total analyte amount.

Typically, the analytes have different electrokinetic mobilities,wherein at least one of the analytes comprises a slowest analyte.Waiting the specified time to inject the analytes into the separationchannel typically involves waiting until the slowest analyte reaches theintersection. By waiting until the slowest analyte has reached theintersection, a representative sample aliquot is obtained such that atotal analyte concentration for the sample is optionally determined,e.g., after separation of the injected sample.

In another aspect, the method of separating two or more analytes anddetermining a total analyte concentration or amount comprises flowing,e.g., electrokinetically or under pressure, at least two analytesthrough a first channel region and through a measurement channel region.The analytes are detected, e.g., unseparated, in the measurement channelregion, thereby determining the total analyte concentration or amount.Detection in the measurement channel typically leads to a signal thatincreases in value until it reaches a constant value, which constantvalue represents the total analyte concentration, e.g., after both slowand fast flowing analytes have reached a detection region. The analytesare then optionally injected into a separation channel, separated, e.g.,electrophoretically, and detected. Concentrations or amounts of eachindividual analyte are optionally determined based on signals detectedafter separation of the analytes. Ratios of the amount of one or more ofthe separated analytes to the total analyte amount or to a portion ofthe total analyte amount are also optionally determined.

In another aspect, the invention provides systems for separating two ormore analytes and determining a total analyte concentration or amount.Such a system typically comprises a microfluidic device comprising abody structure having a plurality of microscale channels disposedtherein. The microscale channels typically comprise a first channelregion for flowing one or more analytes, and a measurement channelregion fluidly coupled to the first channel region. The measurementchannel is used to obtain a total analyte concentration, e.g., after allanalytes have had time to reach a detection channel region. Typicallythe devices include a separation channel fluidly coupled to the firstchannel region and two detection regions. A first detection region istypically positioned proximal to the measurement channel region and asecond detection region is typically positioned proximal to theseparation channel. The first detection region is typically used todetect the total analyte concentration or amount and the seconddetection region is typically used to detect individual analytes, e.g.,after separation.

The system further comprises a fluid direction system fluidly coupled tothe microfluidic device. The fluid direction system directs movement ofthe analytes through the first channel region; movement of the analytesfrom the first channel region into the measurement channel region;movement of the analytes through the first detection region; movement ofthe analytes from the first channel into the separation channel; and,movement of the separated analytes through the second detection region.The fluid direction system typically comprises one or more fluid controlelements, such as pressure sources or electrokinetic controllers,fluidly coupled or air-coupled to the plurality of microscale channels.

A detection system is also included in the systems of the invention todetect total analyte sample aliquots and separated analytes. A detectionsystem is typically positioned proximal to one or more of the firstdetection region or the second detection region. The detection systemdetects the analytes in the first detection region, resulting in a totalanalyte signal corresponding to the total analyte concentration oramount. The detection system also detects the separated analytes in thesecond detection region, resulting in two or more analyte signals, whichtwo or more analyte signals correspond to the two or more separatedanalytes. The detection system optionally comprises a single detectorpositioned proximal to the first detection region and the seconddetection region or a first detector positioned proximal to the firstdetection region and a second detector positioned proximal to the seconddetection region.

Systems of the invention also optionally comprise a computer operablycoupled to the detection system. The computer receives the total analytesignal and the two or more analyte signals. The computer typicallycomprises software comprising at least a first instruction set and asecond instruction set. The instruction sets typically determine thetotal analyte concentration or amount from the total analyte signal andthe concentration or amount of each individual analyte from the two ormore analyte signals. Additional instruction sets are optionallyprovided to sum the individual analyte signals, thus determining thetotal analyte concentration or amount and/or determine a ratio of theamount of one or more analyte to the total analyte amount or to aportion of the total analyte amount.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Schematic illustration of a microfluidic channel configurationoptionally used to determine total analyte concentration.

FIG. 2: Schematic illustration of a microfluidic channel configurationcomprising an alternative intersection or junction for use indetermining total analyte concentration.

FIG. 3: Schematic illustration of a microfluidic channel configurationcomprising a measurement channel for determination of total analyteconcentration.

DETAILED DISCUSSION OF THE INVENTION

The present invention provides methods of providing total analytequantitation on a sample. For example, a sample comprising a mixture ofanalytes, e.g., proteins or nucleic acids, has a total analyteconcentration and an individual analyte concentration for each analytein the mixture. In one embodiment, the present invention provides arepresentative aliquot of the sample. The representative aliquot isseparated and the individual signal peak areas are summed to provide thetotal analyte concentration. In a second embodiment, a sample is flowedthrough a measurement channel region and detected in an unseparatedformat.

When a representative sample aliquot is separated, the individualanalyte signal peak areas are optionally summed and normalized, e.g.,for migration time, to provide the total analyte concentration. Arepresentative sample aliquot is obtained, e.g., in a microfluidicdevice under electrokinetic flow control, by waiting a specified timefor both slow and fast moving analytes to move into the separationregion. The present invention provides, e.g., methods for performing anelectrokinetic separation on a representative aliquot to obtain a totalanalyte concentration or amount.

Prior to separation, total analyte concentration is optionally performedby detecting a sample until a constant value is achieved. For example, asample is flowed through a microfluidic channel, e.g.,electrokinetically such that fast and slow analytes move at differentvelocities. The signal value typically increases as the amount ofanalytes reaching the detector increases. Once a steady signal isobserved, it provides an indication that a representative sample isbeing detected, e.g., both slow and fast moving analytes have reachedthe detector. Once a constant signal value is observed, the totalanalyte concentration is typically determined from the steady statesignal.

In the present invention, a “mixture of analytes” refers to acombination, known or unknown, of biological components or analytes,e.g., proteins, enzymes, polypeptides, carbohydrates, nucleic acids,polynucleotides, or the like. The analytes can be in a complex mixture,such as blood, serum, cell extracts, or in a purified solution, such asa buffered solution of polypeptides or polynucleotides. Typical analytesinclude, but are not limited to, ribosomal RNA or messenger RNA, forwhich a ratio as well as a summation is used to determine concentration.Typically, the components are separated in channels of a microfluidicdevice.

Concentrations or amounts of the individual separated analytes areoptionally determined after detection, e.g., based on signal area orheight. The present invention provides methods of determining the totalamount or concentration of analyte, e.g., the entire amount of nucleicacid in a sample, e.g., a blood or serum sample. The “total analyteconcentration or amount” refers to the entire quantity or concentrationof analytes or other components in a sample or the sum of the quantitiesof all of the individual components or analytes. For example, a samplecomprising various nucleic acids, nucleic acid fragments, and the likehas a total nucleic acid concentration or amount and a concentration oramount for each nucleic acid. Amounts, e.g., absolute amounts, areobtained by measuring the entire quantity present in the tested ormetered sample aliquot and concentrations are measured by determiningthe amount of analyte present in a particular volume of sample, e.g.,the volume of sample that is flowed past the detector.

A general description of analyte separation techniques relevant to thepresent invention, e.g., in microfluidic devices, is provided below. Thedetermination of total analyte quantitation methods is then provided,e.g., using a representative separated sample or an unseparated sample,e.g., in a measurement channel. Systems and devices for performinganalyte separations and total analyte quantitations, e.g., incombination are also described. Additional features will be apparentupon complete review.

I. Separation of Analytes

The samples, e.g., mixtures of analytes, in the present invention aretypically separated in a separation region or separation channel of amicrofluidic device. A sample, e.g., to be assayed, tested, orseparated, is introduced into a first channel of the device, e.g., anintroduction channel, under pressure or electrokinetic control. Whenpressure driven flow, e.g., from a vacuum source or electroosmotic pump,is used, the sample that is introduced typically has no electrokineticbias associated with it. No charge is applied and therefore themolecules or analytes do not move or separate based on charge. Movementof the analytes is due to the application of pressure, as opposed to theapplication of charge. Therefore, two analytes, e.g., unhinderedanalytes not in a sizing gel matrix, with different charges and massesmove in the same direction at substantially the same velocity. However,when electrokinetic driven flow is used to introduce analytes into, orflow analytes through, a channel, the analytes move through the channelwith an electrokinetic bias, i.e., depending on mass to charge ratio.Therefore, some analytes will reach the end of the channel or aparticular intersection before others.

From the first channel or introduction channel, a sample aliquot isinjected, e.g., by cross-injection, into a separation channel. Thecross-injection injects the sample aliquot at the intersection of thefirst channel and the separation channel into the separation channel.Electrokinetic forces typically control fluid flow in separationchannels, e.g., to obtain electrophoretic separation of components oranalytes based on mass/charge ratio or size difference. Typically,electrophoretic separation is used to separate the analytes in themixture, e.g., to separate nucleic acid fragments of various lengths.Electrophoretic separation is the separation of substances achieved byapplying an electric field to samples in a solution or gel. In itssimplest form, it depends on the different velocities with which thesubstances or components move in the field. The velocities depend, e.g.,on the charge and size of the substances, as well as the solution or gelused in the channel.

The separation channels or regions typically comprise a separationmatrix. When the sample is flowed through the separation matrix, thecomponents are separated, e.g., based on physical or chemicalproperties, such as molecular weight and/or charge. The separationmatrix comprises, e.g., a polymer, a gel, a polymer solution, particles,coated surfaces, or the like.

Preferably, the channel, such as separation channel 110 in FIG. 1, is apolyacrylamide gel filled channel, or apolydimethylacrylamide/co-acrylic acid polymer filled channel on whichthe mixture of analytes is electrophoretically separated based on massto charge ratio or molecular weight, when all molecules are similarlycharged. Polyacrylamide used as a separation matrix in a microfluidicchannel is optionally cross-linked or non-cross-linked. Preferably it islinear polyacrylamide, i.e., polydimethylacrylamide orpolydimethylacrylamide/co-acrylic acid. Other possible polymers utilizedinclude cellulose, agarose, and the like. If the components are detectedas they exit the separation region, the components are optionallyidentified by their retention times.

Other gel electrophoretic media that are optionally placed in aseparation channel or region of the invention include silica gels suchas Davisil Silica, E. Merck Silica Gel, Sigma-Aldrich Silica Gel (allavailable from Supelco) in addition to a wide range of silica gelsavailable for various purposes as described in the Aldrichcatalogue/handbook (Aldrich Chemical Company, Milwaukee, Wis.).Preferred gel materials include agarose based gels, various forms ofacrylamide based gels (reagents available from, e.g., Supelco, SIGMA,Aldrich, Sigma-Aldrich and many other sources), colloidal solutions,such as protein colloids (gelatins) and hydrated starches. For a reviewof electrophoretic separation techniques and polyacrylamide gels, see,e.g., The Encyclopedia of Molecular Biology, Kendrew (ed.) (1994); and,Gel Electrophoresis of Proteins: A Practical Approach, 2^(nd) editionHames and Rickwood (Eds.) IRL Press, Oxford England, (1990).

Other types of separation matrices are also optionally used anddiscussed in U.S. patent application Ser. No. 09/093,832 filed Jun. 8,1998, entitled “Microfluidic Matrix Localizations Apparatus andMethods,” by Burd Mehta and Kopf-Sill. Alternate separation matrix mediainclude low pressure chromatography media, such as non-ionicmacroreticular and macroporous resins which adsorb and releasecomponents based upon hydrophilic or hydrophobic interactions, e.g.,Amberchrom and Amberlite resins (available from Supelco), Dowex, andDuolite (all available from Supelco). Other optional media includeaffinity media for purification and separation, such as acrylic beads,agarose beads, cellulose, sepharose, or the like. In addition, a widevariety of resins and chromatography media are also available, e.g.,from Supelco, Sigma, Aldrich, or the like, for example, biotin resins,dye resins, aluminas, carbopacks, and the like. For a review ofchromatography techniques and media, see, e.g., AffinityChromatography—A Practical Approach, Dean et al., (Eds.) IRL Press,Oxford (1985); and, Chromatographic Methods, 5^(th) Edition, Braithwaiteet al., (1996).

For example, a processed protein sample, e.g., that has been desaltedand denatured in SDS, is optionally electrophoresed in a linearpolyacrylamide gel filled separation channel containing SDS to separatethe proteins on the basis of molecular weight of the protein subunits. Adetector is optionally positioned so that it detects the proteins thatare stained in the gel with a fluorescent protein stain. The retentiontime of the proteins as they are electrophoresed through the gel is usedwith markers to measure the molecular weight of the proteins. The areaunder the curve or peak height for each protein is used to determine theconcentration and/or amount of that protein. The present inventionprovides methods for determining the concentration of all proteinscombined in addition to the individual protein concentrations.

Separation of the analytes produces a plurality of signals upondetection. Each signal corresponds to an individual analyte and istypically used to determine the concentration or amount of each analytepresent in the sample (e.g., using the signal peak areas), and/or toidentify the analyte (e.g., using the retention times). The total amountof analyte is optionally obtained by summing or adding the normalized,e.g., peak areas vs. migration velocity peak areas or heights of thevarious analyte signals. However, to obtain an accurate total analytequantitation, a representative sample aliquot, e.g., containing bothslow and fast moving analytes, is typically used, e.g., injected intothe separation channel from the first channel. If a non-representativealiquot is injected, the total analyte concentration will be biased,e.g., for fast moving analytes that reach the intersection of the firstchannel and the separation channel before the slow moving analytes (andtherefore are preferentially injected into the separation channel). Thepresent invention provides methods for accurate total analytequantitation as described below.

II. Determination of Total Analyte Concentration

Analytes are moved through channels, e.g., in microfluidic devices, bythe application of pressure to the analytes, the application of anelectrokinetic gradient, or combinations thereof. The application of theelectrokinetic gradient to flow analytes through a channel introduces anelectrokinetic bias to the analytes, resulting in slower and fastermoving analytes. The present invention provides methods of obtainingrepresentative sample aliquots, e.g., that overcome electrokinetic bias,that are subsequently used to determine total analyte concentration.Electrokinetic bias is overcome by waiting an appropriate time so thatthe detected sample aliquot contains representative amounts of eachindividual analyte, both slow and fast moving analytes. The waiting timeis used because an initial sample aliquot, e.g., the first samplealiquot or portion to reach the detection region, contains faster movinganalytes and very few of the slowest moving analytes. A sample aliquotdetected or injected after the time required for the slowest analyte toreach the detection region or cross-injection region contains theslowest and fastest moving analytes from a continuously flowing sample,thereby providing a representative sample aliquot. For example, when asample is introduced into an introduction channel with electrokineticbias, a cross-injection is typically timed to insure that the slowestanalyte is included in the injected volume, thereby obtaining arepresentative sample. Alternatively, unseparated analytes are detectedas they flow through a detection region. Initially, the signal peak areaincreases as more and more analytes reach the detection region.Eventually the slower analytes reach the detection region and fastanalytes are still being flowed through so the signal peak value becomesconstant. A constant or steady state signal value indicates that arepresentative sample comprising slower and faster moving analytes isflowing past the detector. When pressure based flow is used, theanalytes typically do not move with electrokinetic bias. Therefore allanalytes may reach the detection region simultaneously providing aconstant signal value representing the total analyte concentration.

A sample, e.g., a mixture of analytes, is typically injected into amicrofluidic device through a first channel such as an introductionchannel, a mixing channel, a cross-channel, or the like. Variousreagents are optionally mixed with the sample, e.g., to perform an assaywhich generates a mixture of products to be separated. Typicallyelectrokinetic, e.g., electrophoretic or electroosmotic, forces are usedto flow the analytes through the channels of the invention. A portion ofthe sample, e.g., a sample aliquot, is then cross-injected into aseparation channel, e.g., the volume of material at the intersection ofthe first channel with a separation channel is injected or flowed intothe separation channel. The mixture of analytes is then typicallyelectrophoretically separated.

For example, in FIG. 1 a sample is optionally flowed from reservoir 125,145, 150, or the like into a first channel, e.g., channel 105. In someembodiments, the sample is flowed continuously for a period of time,e.g., until depleted, through the channels of the invention. When thesample flows across the intersection of channel 105 and separationchannel 110, a sample aliquot or the portion of sample in theintersection is cross-injected into separation channel 110, e.g., byapplying a voltage gradient between reservoir 135 and reservoir 140.

In other embodiments, the intersection comprises a split intersectionsuch that a larger volume of sample is injected into the separationchannel. For example, see FIG. 2. In FIG. 2, a sample is flowed througha first channel that is divided into two portions, e.g., channel region205 and channel region 206. The sample is flowed through channel region205, through intersection region 280, and then into channel region 206.A cross injection into separation channel 210, e.g., achieved by avoltage gradient between reservoirs 235 and 240, injects the materialwithin intersection region 280 into separation channel 210. A largervolume of fluid, e.g., the sample aliquot, is thereby injected intoseparation channel 210 from intersection region 280 than is injectedinto separation channel 110 from intersection 180 (as illustrated inFIG. 1).

The samples are typically flowed, e.g., continuously, for a period oftime from the sample reservoirs, e.g., from reservoir 225 in FIG. 2,into the channels, e.g., channel 205. Therefore, when electrokineticforces are applied to the channels, the samples and/or analytes movewith an electrokinetic bias, e.g., analytes with varying mass to chargeratios move at different velocities through the channels. The fastermoving analytes approach the intersection before the slower movinganalytes, but after a period of time, the slower moving analytes alsoapproach the intersection. Since the sample is continuously flowed, thefaster analytes and the slower analytes will both be present at theintersection after specified waiting time, e.g., the time for the sloweranalytes to reach the intersection. The specified time is at least aslong as the time for the slowest analyte to reach the intersection ofthe first channel and the separation channel and possibly longer.

The specified time is optionally determined using markers, e.g.,fluorescent markers that flow faster and/or slower than all analytespresent in the sample. The markers are optionally detected to determineif all analytes of interest were included in the sample aliquot injectedinto the separation channel. If both slow and fast moving markers aredetected, the data is then optionally used to provide a total analyteconcentration measurement. In other embodiments, the time for theslowest analyte is optionally measured in an initial experiment usingthe same sample and then that time is selected as the specified time. Inother embodiments, the analytes are known analytes for which flow ratesat specified voltages are known or can be determined prior to totalanalyte quantitation. The specified waiting time is determined from theflow rate of the slowest moving analyte.

The method typically comprises detecting the signals from the separatedanalytes, which provide an indication of analyte concentrations, therebyoptionally determining the concentration of each individual analyte. Theconcentration or amount is based upon the area under the curve of thepeak detected for each analyte. The peak area is typically determined byintegration of the area under the curve, e.g., using a digitalelectronic integrator. Alternative methods of determining peak areainclude, but are not limited to, cutting out, e.g., with scissors, thearea concerned and weighing it, using a bar graph to approximate thearea under the curve, and by counting squares, e.g., on graph paper onwhich the signal has been plotted. The areas of the peaks are correlatedto the concentrations or amounts of analytes using standards, eitherinternal or external, of known concentration or amount by methods wellknown to those of skill in the art. If the amount is desired, the amountof fluid passing the detector is also measured. Peak heights are alsooptionally used to determine concentration if separation conditions,e.g., temperature, flow rate, and the like, do not alter peak widthsduring separation.

To determine the total analyte concentration or amount, the area or peakheight corresponding to each analyte or the amount of each analyte isnormalized, e.g., time corrected, and summed. Because the sample aliquotinjected into the separation channel is a representative or true samplealiquot, the summation provides a total analyte concentration. In otherwords, the sample aliquot comprises both fast and slow moving analytesbecause the injection into the separation channel is appropriately timedto contain all components of the sample. The total analyte amount orconcentration is optionally used to determine a ratio of one or moreparticular analyte to the total.

Measurement Channels

A “measurement channel” or “loading channel,” as used herein, refers toa channel or channel region that is used to obtain a measurement on asample, e.g., by detecting the sample or a portion of the sample withinthe channel. For example, a sample is flowed through a measurementchannel and detected, e.g., by fluorescence. The fluorescent signalobtained is then optionally analyzed, e.g., to determine sampleconcentration and/or identity.

FIG. 3 provides a schematic illustration of a microfluidic device with ameasurement channel. A sample is typically flowed, e.g., continuouslyfor a period of time, through a first channel, e.g., channel 305, from areservoir, e.g., reservoir 325, 345, 350, 355, 360, or the like. Atintersection 382, the sample stream is typically split and a firstportion flows into measurement channel 315 and a second portioncontinues to flow through channel 305. The portion in measurementchannel 315 is detected in detection region 320. The second portion ofthe sample flows through intersection 380, at which point, the volume ofsample at intersection 380 is cross-injected into separation channel310. The measurement channel is optionally a separate channel or aportion of a channel, e.g., a portion of channel 305. The measurementchannel is optionally downstream or upstream of the separation channeland a device may contain multiple measurement channels. Measurementchannels optionally serve as pre-load channels to test multiple samples.One sample is optionally separated in separation channel 310 whileanother sample is preloaded into measurement channel 315. For example, afirst sample is optionally loaded or flowed from reservoir 345 intofirst channel 305, and into measurement channel 315, where the totalanalyte concentration is determined. The first sample is also flowedinto separation channel 310 for separation and detection of eachcomponent. While the first sample is being separated, a second sample isoptionally loaded into measurement channel 315, e.g., from reservoir350. During this load or preload, the total analyte concentration isdetermined and then the second sample is also separated in channel 310and so forth. The multiple reservoirs are optionally replaced by asipper capillary that is fluidly coupled during operation to, e.g., amicrowell plate comprising a plurality of samples that are sequentiallyloaded and analyzed. Multiple separation channels and measurementchannels are optionally combined into one device to increase samplethroughput even more. For example, using the measurement channel todetermine the total analyte concentration typically allows theseparation to be performed without waiting the specified time andtherefore allows a higher throughput.

Once the analytes are flowed into a measurement channel or region, theanalytes are detected. When flowed into a measurement channel, theanalytes are typically unseparated analytes. As the sample, e.g.,mixture of analytes, flows past a detection region proximal to, orwithin, the measurement channel, the analytes are detected. Whenelectrokinetic flow control is used, the analytes will flow towards thedetection region with electrokinetic bias as discussed above. A signalfrom the analytes, e.g., an absorbance signal, a fluorescence orchemiluminescence signal, a signal due to intercalating dyes, isdetected, e.g., continuously, as the sample is flowed through thedetection region. As the faster analytes approach the detection region,a signal is observed. The sample is typically continuously flowed towardthe detection region and as more and more analytes reach the detectionregion, the signal height or area increases, e.g., the analyte signalvalue increases as more of the slower analytes begin to reach thedetection region. The value of the signal peak area or height reaches aconstant or unchanging value when all of the analytes have reached thedetection region. The constant signal value represents the total analyteamount. The faster analytes are still present in the sample when theslow analytes reach the detection region because the sample is typicallyflowed continuously, i.e., flowed for a period of time and then anothersample is flowed for a period of time, and so forth. Therefore, the fastanalytes are still flowing through the channel when the slowest analytesreach the intersection. Therefore when the peak height or peak areareaches a constant or steady state value, that value corresponds to thetotal analyte concentration or amount, which is determined from areaunder the curve as described above.

The sample is also optionally flowed towards the separation channel,simultaneously or sequentially, to separate a sample aliquot into itsvarious components. The components are detected and their concentrationsdetermined as described above. The individual analyte concentrations areoptionally used to determine one or more ratio or difference ofindividual analyte to total analyte concentration or amount. Inaddition, the signals from each individual analyte may be summed asdescribed above to provide a duplicate measurement of the total analyteconcentration if a representative sample aliquot was used. The two typesof measurements are thus used alone or in combination to provide analyteseparation and total analyte quantitation. Alternatively, total analytequantitation techniques are performed without separation of theanalytes.

III. Systems for Determining Total Analyte Concentration

In the present invention, mixtures of analytes are separated anddetected and total analyte concentrations are determined. For example, amixture of nucleic acids is optionally separated into its variouscomponents which are each quantitated based on the area of the detectedsignals. In addition, the total amount or concentration of all thenucleic acids in a sample is determined by methods described herein.Depending on the detected signal measurements, decisions are optionallymade regarding subsequent fluidic operations, e.g., whether to assay aparticular component in detail to determine, e.g., kinetic information.

The systems described herein generally include microfluidic devices inconjunction with additional instrumentation for controlling fluidtransport, flow rate and direction within the devices, detectioninstrumentation for detecting or sensing results of the operationsperformed by the system, processors, e.g., computers, for instructingthe controlling instrumentation in accordance with preprogrammedinstructions, receiving data from the detection instrumentation, and foranalyzing, storing and interpreting the data, and providing the data andinterpretations in a readily accessible reporting format.

Devices

The microfluidic devices of the present invention are used to obtainmeasurements of total analyte concentration, e.g., in combination withanalyte separation. For example, a mixture of analytes is separated,e.g., electrokinetically, into its individual analytes in a separationchannel and the total amount of analyte in the mixture is determined,e.g., from a summation of individual analyte peaks or from a totalanalyte signal. The present devices are configured to provide separationchannels and measurement channels to obtain total analyte quantitationand separation of analyte peaks.

The devices generally comprise a body structure with microscale channelsfabricated therein. For example, a system of the present inventiontypically comprises, e.g., an introduction channel and a separationchannel. The sizes of the channels are optionally configured to providegood resolution for separations performed in the separation channel.See, e.g., U.S. Ser. No. 60/161,710, by Jaffe et al., entitled “PressureInduced Reagent Introduction and Electrophoretic Separation,” describingthe integrated use of shallow channels for electrokinetic separationsand deep channels for pressure based manipulations, such as sampleintroduction and/or mixing. The introduction and separation channels arefluidly coupled to each other and to various reservoirs or other sourcesof materials. For example, the two channels typically meet or cross toprovide an intersection. Alternatively, the introduction channel is asplit channel such that the intersection is formed by three channelregions, the two parts of the introduction channel and the separationchannel. “Intersection,” as used herein, refers to any type of fluidconnection between two or more channels. Typical channel intersectionsare illustrated by intersection 180 in FIG. 1 and intersection region280 in FIG. 2. Materials are typically electrokinetically loaded andinjected from an introduction channel into a separation channel. Forexample, a cross-injection from an introduction channel into aseparation channel injects the volume of fluid at the intersection ofthe introduction channel and the separation channel into the separationchannel. Cross-injections are typically floating injections or pinchedinjections as described in Ramsey, WO96/04547. For example, in afloating cross-injection, the voltages across the introduction orloading channel are turned off or allowed to float and a voltage isapplied across the separation channel. This allows the fluid at theintersection to flow into the separation channel and allows some portionof fluid from the introduction channel to flow into the separationchannel as well. In a pinched injection, the voltages across theintroduction or loading channel are adjusted to minimize or eliminateleakage, while a voltage is applied across the separation channel thusinjecting a plug of fluid from the intersection into the separationchannel.

Optionally, the separation channel is a gel filled channel, e.g., alinear polyacrylamide gel filled channel or a polymer solution filledchannel, e.g., a polyacrylamide polymer solution or apolydimethylacrylamide/co-acrylic acid polymer, that separates thevarious components based on molecular weight, wherein each component iseluted from the separation channel with a different retention time. Thecomponents are then optionally detected and their molecular weightsdetermined, e.g., by the retention time. In addition, the concentrationsand/or amounts of each component are optionally determined based on peakarea, peak height, or the like.

A measurement channel is also optionally included in the microfluidicdevices and systems in the present invention. A measurement channeltypically intersects an introduction channel, but is optionally a regionof the introduction channel. The position of the measurement channel isoptionally downstream or upstream from the intersection of theseparation channel and the introduction channel and multiple measurementchannels can be used, e.g., to measure the total analyte concentration,as described above, in multiple samples simultaneously. FIG. 3illustrates a typical measurement channel, e.g., measurement channel315. The use of measurement channels is described above.

Detection regions are also included in the present devices. Thedetection region is optionally a subunit of a channel, such as detectionregion 120 in FIG. 1. Alternatively, the detection region optionallycomprises a distinct channel that is fluidly coupled to the plurality ofchannels in the microfluidic device. The detection region is optionallylocated anywhere along the length of the separation channel or region.For example, a detection region located at the most downstream point orend of a separation channel detects the separated components as theyexit the separation channel. Detection regions are also typicallyincluded in the measurement channels of the invention, for detection ofunseparated analytes to determine a total analyte concentration. In someembodiments, a single detector is used that is positioned proximal toboth a separation channel and a measurement region, e.g., proximal todetection regions located within each channel.

The detection window or region at which a signal is monitored typicallyincludes a transparent cover allowing visual or optical observation anddetection of the assay results, e.g., observation of a colorimetric orfluorometric signal or label. Examples of suitable detectors are wellknown to those of skill in the art and are discussed in more detailbelow.

Reservoirs, e.g., for storing, discarding, or supplying, samples,analytes, reagents, buffers, and the like, are also optionally includedin the devices of the present invention. For example, a reservoir for asample or a sample well is optionally located at one end of anintroduction channel for introduction of the sample into theintroduction channel. The reservoirs are the locations or wells at whichsamples, components, reagents, and the like are added into the devicefor assays and/or separations to take place. Introduction of theseelements into the system is carried out as described below.

Pressure sources are also optionally applied at the reservoirs of theinvention. Typically, channels, such as channel 105 in FIG. 1, connectthe reservoirs to a pump or other pressure source(s). For example avacuum source may be fluidly coupled to the device at a waste reservoirlocated at the end of a channel, e.g., reservoir 130 at the downstreamend of channel 105 in FIG. 1. The vacuum source draws fluid into thechannel, e.g., for mixing or reacting with other reagents. Additionally,the vacuum optionally draws any excess or unused material, e.g.,material not injected into the separation channel, into a wastereservoir to which the vacuum source is fluidly coupled. Alternatively,a positive pressure source is fluidly coupled to a sample well orreservoir at one end of a channel. The pressure then forces the materialinto and through the channel. Pressures on the separation channels areoptionally adjusted, e.g., individually, to avoid flowing a sample froma separation channel into the waste well.

Electrokinetic forces, e.g., high or low voltages or currents, are alsooptionally applied at reservoirs to the materials in the channels. Forexample, voltage gradients applied across a separation channel are usedto move fluid down the channel, thus separating the components of thematerial as they move through the channel at different rates.

Various channel configuration embodiments of the present systems aredescribed above and shown in FIGS. 1, 2, and 3. The channelconfigurations given above are examples of possible configurations.However, various configurations and dimensions are possible toaccommodate the methods described herein. In fact, a variety ofmicroscale systems are optionally adapted to the present invention byincorporating varied channels depths, lengths, separation gels, particlesets, and the like. These devices are described in various PCTapplications and issued U.S. Patents by the inventors and theircoworkers, including U.S. Pat. No. 5,699,157 (J. Wallace Parce) issuedDec. 12, 1997, U.S. Pat. No. 5,779,868 (J. Wallace Parce et al.) issuedJul. 7, 1998, U.S. Pat. No. 5,800,690 (Calvin Y. H. Chow et al.) issuedSep. 1, 1998, U.S. Pat. No. 5,842,787 (Anne R. Kopf-Sill et al.) issuedDec. 1, 1998, U.S. Pat. No. 5,852,495 (J. Wallace Parce) issued Dec. 12,1998, U.S. Pat. No. 5,869,004 (J. Wallace Parce et al.) Feb. 9, 1999,U.S. Pat. No. 5,876,675 (Colin B. Kennedy) issued Mar. 2, 1999, U.S.Pat. No. 5,880,071 (J. Wallace Parce et al.) issued Mar. 9, 1999, U.S.Pat. No. 5,882,465 (Richard J. McReynolds) issued Mar. 16, 1999, U.S.Pat. No. 5,885,470 ( J. Wallace Parce et al.) issued Mar. 23, 1999, U.S.Pat. No. 5,942,443 (J. Wallace Parce et al.) issued Aug. 24, 1997 U.S.Pat. No. 5,948,227 (Robert S. Dubrow) issued Sep. 7, 1999, U.S. Pat. No.5,955,028 (Calvin Y. H. Chow) issued Sep. 21, 1999, U.S. Pat. No.5,957,579 (Anne R. Kopf-Sill et al.) issued Sep. 28, 1999, U.S. Pat. No.5,958,203 (J. Wallace Parce et al.) issued Sep. 28, 1999, U.S. Pat. No.5,958,694 (Theo T. Nikiforov) issued Sep. 28, 1999, U.S. Pat. No.5,959,291 (Morten J. Jensen) issued Sep. 28, 1999, U.S. Pat. No.5,964,995 (Theo T. Nikiforov et al.) issued Oct. 12, 1999, U.S. Pat. No.5,965,001 (Calvin Y. H. Chow et al.) issued Oct. 12, 1999, U.S. Pat. No.5,965,410 (Calvin Y. H. Chow et al.) issued Oct. 12, 1999, U.S. Pat. No.5,972,187 (J. Wallace Parce et al.) issued Oct. 26, 1999, U.S. Pat. No.5,976,336 (Robert S. Dubrow et al.) issued Nov. 2, 1999, U.S. Pat. No.5,989,402 (Calvin Y. H. Chow et al.) issued Nov. 23, 1999, U.S. Pat. No.6,001,231 (Anne R. Kopf-Sill) issued Dec. 14, 1999, U.S. Pat. No.6,011,252 (Morten J. Jensen) issued Jan. 4, 2000, U.S. Pat. No.6,012,902 (J. Wallace Parce) issued Jan. 11, 2000, U.S. Pat. No.6,042,709 (J. Wallace Parce et al.) issued Mar. 28, 2000, U.S. Pat. No.6,042,710 (Robert S. Dubrow) issued Mar. 28, 2000, U.S. Pat. No.6,046,056 (J. Wallace Parce et al.) issued Apr. 4, 2000, U.S. Pat. No.6,048,498 (Colin B. Kennedy) issued Apr. 11, 2000, U.S. Pat. No.6,068,752 (Robert S. Dubrow et al.) issued May 30, 2000, U.S. Pat. No.6,071,478 (Calvin Y. H. Chow) issued Jun. 6, 2000, U.S. Pat. No.6,074,725 (Colin B. Kennedy) issued Jun. 13, 2000, U.S. Pat. No.6,080,295 (J. Wallace Parce et al.) issued Jun. 27, 2000, U.S. Pat. No.6,086,740 (Colin B. Kennedy) issued Jul. 11, 2000, U.S. Pat. No.6,086,825 (Steven A. Sundberg et al.) issued Jul. 11, 2000, U.S. Pat.No. 6,090,251 (Steven A. Sundberg et al.) issued Jul. 18, 2000, U.S.Pat. No. 6,100,541 (Robert Nagle et al.) issued Aug. 8, 2000, U.S. Pat.No. 6,107,044 (Theo T. Nikiforov) issued Aug. 22, 2000, U.S. Pat. No.6,123,798 (Khushroo Gandhi et al.) issued Sep. 26, 2000, U.S. Pat. No.6,129,826 (Theo T. Nikiforov et al.) issued Oct. 10, 2000, U.S. Pat. No.6,132,685 (Joseph E. Kersco et al.) issued Oct. 17, 2000, U.S. Pat. No.6,148,508 (Jeffrey A. Wolk) issued Nov. 21, 2000, U.S. Pat. No.6,149,787 (Andrea W. Chow et al.) issued Nov. 21, 2000, U.S. Pat. No.6,149,870 (J. Wallace Parce et al.) issued Nov. 21, 2000, U.S. Pat. No.6,150,119 (Anne R. Kopf-Sill et al.) issued Nov. 21, 2000, U.S. Pat. No.6,150,180 (J. Wallace Parce et al.) issued Nov. 21, 2000, U.S. Pat. No.6,153,073 (Robert S. Dubrow et al.) issued Nov. 28, 2000, U.S. Pat. No.6,156,181 (J. Wallace Parce et al.) issued Dec. 5, 2000, U.S. Pat. No.6,167,910 (Calvin Y. H. Chow) issued Jan. 2, 2001, U.S. Pat. No.6,171,067 (J. Wallace Parce) issued Jan. 9, 2001, U.S. Pat. No.6,171,850 (Robert Nagle et al.) issued Jan. 9, 2001, U.S. Pat. No.6,172,353 (Morten J. Jensen) issued Jan. 9, 2001, U.S. Pat. No.6,174,675 (Calvin Y. H. Chow et al.) issued Jan. 16, 2001, U.S. Pat. No.6,182,733 (Richard J. McReynolds) issued Feb. 6, 2001, and U.S. Pat. No.6,186,660 (Anne R. Kopf-Sill et al.) issued Feb. 13, 2001; and publishedPCT applications, such as, WO 98/00231, WO 98/00705, WO 98/00707, WO98/02728, WO 98/05424, WO 98/2281 1, WO 98/45481, WO 98/45929, WO98/46438, and WO 98/49548, WO 98/55852, WO 98/56505, WO 98/56956, WO99/00649, WO 99/10735, WO 99/12016, WO 99/16162, WO 99/19056, WO99/19516, WO 99/29497, WO 99/31495, WO 99/34205, WO 99/43432, WO99/44217, WO 99/56954, WO 99/64836, WO 99/64840, WO 99/64848, WO99/67639, WO 00/07026, WO 00/09753, WO 00/10015, WO 00/21666, WO00/22424, WO 00/26657, WO 00/42212, WO 00/43766, WO 00/45172, WO00/46594, WO 00/50172, WO 00/50642, WO 00/58719, WO 00/060108, WO00/070080, WO 00/070353, WO 00/072016, WO 00/73799, WO 00/078454, WO00/102850, and WO 00/114865.

For example, pioneering technology providing cell based microscaleassays are set forth in U.S. Pat. No. 5,942,443, by Parce et al. “HighThroughput Screening Assay Systems in Microscale Fluidic Devices” and,e.g., in Ser. No. 60/128,643 filed Apr. 4, 1999, entitled “Manipulationof Microparticles In Microfluidic Systems,” by Mehta et al. Completeintegrated systems with fluid handling, signal detection, sample storageand sample accessing are available. For example, U.S. Pat. No. 5,942,443provides pioneering technology for the integration of microfluidics andsample selection and manipulation.

The devices described above are used in the present invention, e.g., toseparate a mixture of analytes, to determine total analyte concentrationor amount, to determine concentration or amount of individual analytes,and the like.

Although the devices and systems specifically illustrated herein aregenerally described in terms of the performance of a few or oneparticular operation, it will be readily appreciated from thisdisclosure that the flexibility of these systems permits easyintegration of additional operations into these devices. For example,the devices and systems described will optionally include structures,reagents and systems for performing virtually any number of operationsboth upstream and downstream from the operations specifically describedherein. Such upstream operations include sample handling and preparationoperations, e.g., cell separation, extraction, purification,amplification, cellular activation, labeling reactions, dilution,aliquotting, and the like. Similarly, downstream operations may includesimilar operations, including, e.g., labeling of components, assays anddetection operations, electrokinetic or pressure-based injection ofcomponents into contact with particle sets, or materials released fromparticle sets, or the like.

Fluid Direction System and Fluid Control Elements

A variety of controlling instrumentation is optionally utilized inconjunction with the microfluidic devices described above, forcontrolling the transport and direction of fluidic materials and/ormaterials within the devices of the present invention, e.g., bypressure-based or electrokinetic control. In general, analytes and/orother components or reagents are flowed in a microscale system byelectrokinetic (including either electroosmotic or electrophoretic)techniques, or by using pressure-based flow mechanisms, or combinationsthereof. In the present system, electrokinetic transport is typicallyused. For example, various analytes are transported through the firstchannel or introduction channel by electrokinetic gradients, whichcauses electrokinetic bias in the sample aliquot injected into theseparation channel unless an appropriate time passes, e.g., to allow theslowest analytes to reach the cross-injection intersection. After theappropriate time, a representative sample aliquot is obtained and atotal analyte concentration is optionally determined.

In the present system, the fluid direction system controls thetransport, flow and/or movement of a sample through the microfluidicdevice. For example, the fluid direction system optionally directs thepressure-based movement of a sample into the device, e.g., through asipper capillary. The fluid direction system also directs electrokineticbased movement of samples, e.g., into and through a separation channel.Electrokinetic based movement though the separation channel results inseparated analytes. In particular, the fluid direction system directsmovement of at least two analytes through the first channel region;movement of the at least two analytes from the first channel region intothe measurement channel region; movement of the at least two analytesthrough the first detection region; movement of the at least twoanalytes from the first channel into the separation channel, therebyproducing two or more separated analytes; and/or, movement of the two ormore separated analytes through the second detection region.

Electrokinetic material transport systems or electrokinetic controllersare used in the present invention to provide movement of analytes,reagents, and the like, through microfluidic channels. “Electrokineticmaterial transport systems,” as used herein, include systems thattransport and direct materials within a microchannel and/or chambercontaining structure, through the application of electrical fields tothe materials, thereby causing material movement through and among thechannel and/or chambers, i.e., cations will move toward a negativeelectrode, while anions will move toward a positive electrode. Forexample, movement of fluids toward or away from a cathode or anode cancause movement of nucleic acids, proteins, enzymes, cells, modulators,etc. suspended within the fluid. Similarly, the components, e.g.,polynucleotides, polypeptides, proteins, antibodies, carbohydrates, etc.can be charged, in which case they will move toward an oppositelycharged electrode (indeed, in this case, it is possible to achieve fluidflow in one direction while achieving particle flow in the oppositedirection). In this embodiment, the fluid can be immobile or flowing andcan comprise a matrix as in electrophoresis. For example, nucleic acidswhich have similar charge/mass ratios are optionally separated based onsize in a channel comprising a size-discriminant separation matrix, suchas polyacrylamide.

Typically, the electrokinetic material transport and direction systemsof the invention rely upon the electrophoretic mobility of chargedspecies within the electric field applied to the structure. Such systemsare more particularly referred to as electrophoretic material transportsystems. For example, in the present system, separation of a mixture ofcomponents into its individual components optionally occurs byelectrophoretic separation. For electrophoretic applications, the wallsof interior channels of the electrokinetic transport system areoptionally charged or uncharged. Typical electrokinetic transportsystems are made of glass, charged polymers, or uncharged polymers. Theinterior channels are optionally coated with a material that alters thesurface charge of the channel.

A variety of electrokinetic controllers and systems which are optionallyused in the present invention are described, e.g., in Ramsey WO96/04547, Parce et al. WO 98/46438 and Dubrow et al., WO 98/49548, aswell as a variety of other references noted herein.

Use of electrokinetic transport to control material movement ininterconnected channel structures was described, e.g., in WO 96/04547and U.S. Pat. No. 5,858,195 by Ramsey. An exemplary controller isdescribed in U.S. Pat. No. 5,800,690. Modulating voltages areconcomitantly applied to the various reservoirs to affect a desiredfluid flow characteristic, e.g., continuous or discontinuous (e.g.,regularly pulsed field(s) causing the sample to oscillate direction oftravel) flow of labeled components in one or more channels toward awaste reservoir. Particularly, modulation of the voltages applied at thevarious reservoirs, such as reservoirs 325, 330, 340, 365, and the likein FIG. 3, can move and direct fluid flow through the interconnectedchannel structure of the device.

Other methods of transport are also available for situations in whichelectrokinetic methods are not desirable. For example, sampleintroduction and reaction are optionally carried out in a pressure-basedsystem and high throughput systems typically use pressure induced sampleintroduction. In addition, cells are desirably flowed usingpressure-based flow mechanisms.

Pressure based flow is also desirable in systems in which electrokinetictransport is also used. For example, pressure based flow is optionallyused for introducing and reacting reagents in a system in which theproducts are electrophoretically separated. Therefore, in the presentinvention, pressure-based systems are optionally combined with theelectrokinetic transport systems described above.

Pressure can be applied to microscale elements to achieve fluid movementusing any of a variety of techniques. Fluid flow (and flow of materialssuspended or solubilized within the fluid, including cells or otherparticles) is optionally regulated by pressure based mechanisms such asthose based upon fluid displacement, e.g., using a piston, pressurediaphragm, vacuum pump, probe, or the like to displace liquid and raiseor lower the pressure at a site in the microfluidic system. The pressureis optionally pneumatic, e.g., a pressurized gas, or uses hydraulicforces, e.g., pressurized liquid, or alternatively, uses a positivedisplacement mechanism, i.e., a plunger fitted into a materialreservoir, for forcing material through a channel or other conduit, oris a combination of such forces. For example, pressure is optionallygenerated via compression of air, in which case air, not liquid, is usedas the coupling medium.

Internal sources of fluid transport include microfabricated pumps, e.g.,diaphragm pumps, thermal pumps, lamb wave pumps, and the like that havebeen described in the art. See, e.g., U.S. Pat. Nos. 5,271,724,5,277,556, and 5,375,979 and Published PCT application Ser. Nos. WO94/05414 and WO 97/02357. Preferably, external pressure sources areused, and applied to ports at channel termini. These applied pressures,or vacuums, generate pressure differentials across the lengths ofchannels to drive fluid flow through them. In the interconnected channelnetworks described herein, differential flow rates on volumes areoptionally accomplished by applying different pressures or vacuums atmultiple ports, or preferably, by applying a single vacuum at a commonwaste port and configuring the various channels with appropriateresistance to yield desired flow rates. Example systems are described inU.S. Ser. No. 09/238,467, filed Jan. 28, 1999.

In some embodiments, a vacuum source is applied to a reservoir or wellat one end of a channel to draw the suspension through the channel. Forexample, a vacuum source is optionally placed at a reservoir in thepresent devices for drawing fluid into a channel, e.g., a vacuum sourceat reservoir 330 in FIG. 3 applies a pressure to channel 305, thusdrawing fluid from reservoir 325, 345, 350, 355, 360, or the like intochannel 305.

Pressure or vacuum sources are optionally supplied external to thedevice or system, e.g., external vacuum or pressure pumps sealablyfitted to the inlet or outlet of the channel, or they are internal tothe device, e.g., microfabricated pumps integrated into the device andoperably linked to the channel. Examples of microfabricated pumps havebeen widely described in the art. See, e.g., published InternationalApplication No. WO 97/02357.

Another alternative to electrokinetic transport is an electroosmoticpump which uses electroosmotic forces to generate pressure based flow.See, e.g., published International Application No. WO 99/16162 by Parce,entitled “Micropump.” An electroosmotic pump typically comprises twochannels. The pump utilizes electroosmotic pumping of fluid in onechannel or region to generate pressure based fluid flow in a connectedchannel, where the connected channel has substantially no electroosmoticflow generated.

Hydrostatic, wicking and capillary forces are also optionally used toprovide pressure for fluid flow of materials such as enzymes,substrates, modulators, or protein mixtures. See, e.g., “METHOD ANDAPPARATUS FOR CONTINUOUS LIQUID FLOW IN MICROSCALE CHANNELS USINGPRESSURE INJECTION, WICKING AND ELECTROKINETIC INJECTION,” by Alajoki etal, U.S. Ser. No. 09/245,627, filed Feb. 5, 1999. In these methods, anadsorbent material or a branched capillary structure is placed influidic contact with a region where pressure is applied, thereby causingfluid to move towards the adsorbent material or branched capillarystructure. The capillary forces are optionally used in conjunction withthe electrokinetic or pressure-based flow in the present invention todraw fluid through the channels, e.g., the measurement channel. Thecapillary action pulls material through a channel. For example a wick isoptionally added to a channel to aid fluid flow by drawing liquidthrough the channel.

Mechanisms for reducing adsorption of materials during fluid-based floware described in U.S. Ser. No. 09/310,027, “PREVENTION OF SURFACEADSORPTION IN MICROCHANNELS BY APPLICATION OF ELECTRIC CURRENT DURINGPRESSURE-INDUCED FLOW” filed May 11, 1999 by Parce et al. In brief,adsorption of cells, components, proteins, nucleic acids, and othermaterials to channel walls or other microscale components duringpressure-based flow can be reduced by applying an electric field such asan alternating current to the material during flow. Such an electriccurrent can cause electrokinetic biasing of the analytes as they flowthrough the channel. For example, in the present invention pressurebased flow is optionally used in combination with an alternating currentto provide fluid flow through the introduction channel or first channel,the measurement channel, or the like. The introduction of electrokineticbias to the samples is compensated for by the methods provided herein.

In an alternate embodiment, microfluidic systems can be incorporatedinto centrifuge rotor devices, which are spun in a centrifuge. Fluidsand particles travel through the device due to gravitational andcentripetal/centrifugal pressure forces.

Typically, the fluid control elements described above are controlledand/or coordinated by controller systems appropriately configured toreceive or interface with a microfluidic device or system element asdescribed herein. For example, the controller and/or detector,optionally includes a stage upon which the device of the invention ismounted to facilitate appropriate interfacing between the controllerand/or detector and the device. Typically, the stage includes anappropriate mounting/alignment structural element, such as a nestingwell, alignment pins and/or holes, asymmetric edge structures (tofacilitate proper device alignment), and the like. Many suchconfigurations are described in the references cited herein.

The controlling instrumentation discussed above is also used to providefor electrokinetic injection or withdrawal of material downstream of theregion of interest to control an upstream flow rate. The sameinstrumentation and techniques described above are also utilized toinject a fluid into a downstream port to function as a flow controlelement.

The above transport and control systems are optionally used in thesystems of the present invention, alone or in combinations, to providefluid flow through the channels of the invention. In addition totransport through the microfluidic system, the invention also providesfor introduction of sample or reagents, e.g., enzymes, proteins,substrates, modulators, and the like, into the microfluidic system.

Sources of Assay Components and Integration With Microfluidic Formats

Reservoirs or wells are provided in the present invention as sources ofsamples, mixtures of analytes, analytes, reagents, buffers, and thelike, or as waste wells. Such wells include, e.g., reservoirs 225, 230,235, and 240 in FIG. 2. For example, a sample is optionally introducedinto the device through reservoir 225.

Sources of samples, e.g., mixtures of analytes such as polynucleotides,polypeptides, and the like, are fluidly coupled to the microchannelsnoted herein in any of a variety of ways. In particular, those systemscomprising sources of materials set forth in Knapp et al. “Closed LoopBiochemical Analyzers” (WO 98/45481; PCT/US98/06723) and Parce et al.“High Throughput Screening Assay Systems in Microscale Fluidic Devices”WO 98/00231 and, e.g., in 60/128,643 filed Apr. 4, 1999, entitled“Manipulation of Microparticles In Microfluidic Systems,” by Mehta etal. are applicable.

In these systems, a “pipettor channel” (a channel in which componentscan be moved from a source to a microscale element such as a secondchannel or reservoir) is temporarily or permanently coupled to a sourceof material. The source can be internal or external to a microfluidicdevice comprising the pipettor channel. Example sources includemicrowell plates, membranes or other solid substrates comprisinglyophilized components, wells or reservoirs in the body of themicroscale device itself and others.

For example, the source of a sample, mixture of components, or buffercan be a microwell plate external to the body structure, having, e.g.,at least one well with the selected cell type or component.Alternatively, samples are contained in a well disposed on the surfaceof the body structure, a reservoir disposed within the body structure; acontainer external to the body structure comprising at least onecompartment comprising the samples, or a solid phase structurecomprising the samples or reagents in lyophilized or otherwise driedform. For example, in FIG. 3, various wells are comprised within thedevice. Reservoirs 325, 345, 350, 355, and 360 each typically comprise adifferent sample. Additional reservoirs are optionally included.

A loading channel region is optionally fluidly coupled to a pipettorchannel with a port external to the body structure. The loading channelcan be coupled to an electropipettor channel with a port external to thebody structure, a pressure-based pipettor channel with a port externalto the body structure, a pipettor channel with a port internal to thebody structure, an internal channel within the body structure fluidlycoupled to a well on the surface of the body structure, an internalchannel within the body structure fluidly coupled to a well within thebody structure, or the like.

The integrated microfluidic system of the invention optionally includesa very wide variety of storage elements for storing samples and reagentsto be assessed. These include well plates, matrices, membranes and thelike. The reagents are stored in liquids (e.g., in a well on amicrotiter plate), or in lyophilized form (e.g., dried on a membrane orin a porous matrix), and can be transported to an array component,region, or channel of the microfluidic device using conventionalrobotics, or using an electropipettor or pressure pipettor channelfluidly coupled to a region or channel of the microfluidic system.

Detectors

Once separated, the components of a sample are typically detected. Inthe present invention, the entire sample, e.g., an unseparated sample,is also optionally detected, e.g., before separation. For example, amixture of polynucleotides is optionally detected to determine a totalanalyte concentration, e.g., in a measurement channel, and separated anddetected in a separation channel. In another embodiment, the sample isseparated using a representative sample aliquot, and the detectedsignals from the separated components are summed to determine totalanalyte concentration. The detector(s) optionally monitors one or aplurality of signals, e.g., from one or more analyte of interest, in aseparation channel or in the measurement channel. For example, thedetector optionally monitors an optical signal, e.g., an analyte signal,that corresponds to a labeled analyte, such as a labeled nucleic acid orpolypeptide, located in a detection region or detection channel, e.g., adetection region that is proximal to or within a separation channel. Inanother embodiment, the detector is positioned at the downstream end ofthe separation region or channel and detects a plurality of signals fromthe separated components as they elute from a separation matrix.

Nucleic acids, proteins, polynucleotides, polypeptides, antibodies, orother components which can emit a detectable signal, e.g., fluoresceinlabeled analytes, are optionally flowed past the detector, or,alternatively, the detector can move relative to the array to examinevarious positions in the array (or, the detector can simultaneouslymonitor a number of spatial positions corresponding to channel regions,e.g., as in a CCD array). In other embodiments, the analytes areunlabeled analytes, e.g., unlabeled nucleic acids or polynucleotides,that are detected using an intercalating dye, e.g., a fluorescent dyethat inserts into a nucleic acid double helix.

The detector typically includes or is operably linked to a computer,e.g., which has software for converting detector signal information intoassay result information, e.g., molecular weight based on retention timeor elution time, identity of a analyte, concentration of an analyte,total analyte concentration, or the like.

Examples of detection systems include optical sensors, temperaturesensors, pressure sensors, pH sensors, conductivity sensors, and thelike. Each of these types of sensors is readily incorporated into themicrofluidic systems described herein. In these systems, such detectorsare placed either within or adjacent to the microfluidic device or oneor more channels, chambers or conduits of the device, such that thedetector is within sensory communication with the device, channel, orchamber. The phrase “proximal,” to a particular element or region, asused herein, generally refers to the placement of the detector in aposition such that the detector is capable of detecting the property ofthe microfluidic device, a portion of the microfluidic device, or thecontents of a portion of the microfluidic device, for which thatdetector was intended. For example, a pH sensor placed in sensorycommunication with a microscale channel is capable of determining the pHof a fluid disposed in that channel. Similarly, a temperature sensorplaced in sensory communication with the body of a microfluidic deviceis capable of determining the temperature of the device itself.

Particularly preferred detection systems include optical detectionsystems for detecting an optical property of a material within thechannels and/or chambers of the microfluidic devices that areincorporated into the microfluidic systems described herein. For examplefluorescent or chemiluminescent detectors are typically preferred. Insome embodiments, absorbance is used to detect, e.g. nucleic acids. Suchoptical detection systems are typically placed adjacent to a microscalechannel of a microfluidic device, and are in sensory communication withthe channel via an optical detection window that is disposed across thechannel or chamber of the device. Optical detection systems includesystems that are capable of measuring the light emitted from materialwithin the channel, the transmissivity or absorbance of the material, aswell as the material's spectral characteristics. In preferred aspects,the detector measures an amount of light emitted from the material, suchas a fluorescent or chemiluminescent material. As such, the detectionsystem will typically include collection optics for gathering a lightbased signal transmitted through the detection window and transmittingthat signal to an appropriate light detector. Microscope objectives ofvarying power, field diameter, and focal length are readily utilized asat least a portion of this optical train. The light detectors areoptionally photodiodes, avalanche photodiodes, photomultiplier tubes,diode arrays, or in some cases, imaging systems, such as charged coupleddevices (CCDs) and the like. In preferred aspects, photodiodes areutilized, at least in part, as the light detectors. The detection systemis typically coupled to a computer (described in greater detail below),via an analog to digital or digital to analog converter, fortransmitting detected light data to the computer for analysis, storageand data manipulation.

In the case of fluorescent materials such as fluorescently labeledintercalating dues used to detect nucleic acids, the detector typicallyincludes a light source which produces light at an appropriatewavelength for activating the fluorescent material, as well as opticsfor directing the light source through the detection window to thematerial contained in the channel or chamber. The light source can beany number of light sources that provides an appropriate wavelength,including lasers, laser diodes and LEDs. Other light sources arerequired for other detection systems. For example, broad band lightsources, e.g., in conjunction with appropriate optical filters to selectwavelength(s) of interest, are typically used in lightscattering/transmissivity detection schemes, and the like. Typically,light selection parameters are well known to those of skill in the art.

The detector can exist as a separate unit, but is preferably integratedwith a controller system, into a single instrument. Integration of thesefunctions into a single unit facilitates connection of these instrumentswith the computer (described below), by permitting the use of few or asingle communication port(s) for transmitting information between thecontroller, the detector and the computer.

Computer

As noted above, either or both of the fluid direction system and/or thedetection system are coupled to an appropriately programmed processor orcomputer which functions to instruct the operation of these instrumentsin accordance with preprogrammed or user input instructions, receivedata and information from these instruments, and interpret, manipulateand report this information to the user. As such, the computer istypically appropriately coupled to one or both of these instruments(e.g., including an analog to digital or digital to analog converter asneeded).

The computer typically includes appropriate software for receiving userinstructions, either in the form of user input into set parameterfields, e.g., in a GUI, or in the form of preprogrammed instructions,e.g., preprogrammed for a variety of different specific operations. Thesoftware then converts these instructions to appropriate language forinstructing the operation of the fluid direction and transportcontroller to carry out the desired operation. For example, the softwareoptionally directs the fluid direction system to transport the sampleinto a first channel of the device or into a separation channel, e.g.,using electrokinetic forces. In addition, the software optionallydirects the fluid direction system to inject or cross inject a samplealiquot from the first channel into the separation channel in which theanalytes are separated. Any other movement necessary to assay, separate,or detect the sample is also optionally directed by the softwareinstructions.

The computer additionally receives the data from the one or moresensors/detectors included within the system, and interprets the data,and either provides it in a user understood format, or uses that data toinitiate further controller instructions in accordance with theprogramming, e.g., such as in monitoring and control of flow rates,temperatures, applied voltages, and the like.

In the present invention, the computer typically includes software forthe monitoring of materials in the channels. Additionally the softwareis optionally used to control electrokinetic or pressure-modulatedinjection or withdrawal of material.

In addition, the computer optionally includes software for deconvolutionof the signal or signals from the detection system. Computer softwareinstruction sets are typically used in the present invention todetermine the total analyte concentration, each individual analyteconcentration, one or more ratios of analyte concentration to totalanalyte concentration, or the like, based on analyte and total analytepeak areas (area under the curve), heights, and/or amplitudes whichcorrespond to the amount of analyte detected (for example, deconvolutionquantitates the amount of each analyte in a sample, the total amount ofanalyte present in a sample and calculates ratios between the two. Theanalyte amounts, e.g., of individual analytes or total analyte, arecalculated, e.g., based on peak area which corresponds to the amount ofmaterial detected). The peak areas are determined, e.g., by integrationof the area under the curve produced by a signal plot. The concentrationis determined by correlating the amount of material detected based onpeak area to the amount of fluid flowed through the detection regionduring detection. For example when a representative sample aliquot of aknown volume (the volume is optionally determined as it flows past thedetector, e.g., into a waste reservoir) is injected into a separationchannel and the separated analytes detected. The signal peakscorresponding to the separated analytes are integrated, e.g., usingcomputer software for integration, to determine the amount of materialdetected. The total amount of material is determined by summing thesignal peak areas and the concentration is determined by dividing theamount of material by the volume of the sample aliquot detected.

Kits

Generally, the microfluidic devices described herein are optionallypackaged to include reagents for performing the device's preferredfunction. For example, the kits can include any of the microfluidicdevices described along with assay components, reagents, samplematerials, particle sets, salts, separation matrices,control/calibrating materials, or the like. Such kits also typicallyinclude appropriate instructions for using the devices and reagents, andin cases where reagents are not predisposed in the devices themselves,with appropriate instructions for introducing the reagents into thechannels and/or chambers of the device. In this latter case, these kitsoptionally include special ancillary devices for introducing materialsinto the microfluidic systems, e.g., appropriately configuredsyringes/pumps, or the like (in one preferred embodiment, the deviceitself comprises a pipettor element, such as an electropipettor forintroducing material into channels and chambers within the device). Inthe former case, such kits typically include a microfluidic device withnecessary reagents predisposed in the channels/chambers of the device.Generally, such reagents are provided in a stabilized form, so as toprevent degradation or other loss during prolonged storage, e.g., fromleakage. A number of stabilizing processes are widely used for reagentsthat are to be stored, such as the inclusion of chemical stabilizers(e.g., enzymatic inhibitors, microbicides/bacteriostats,anticoagulants), the physical stabilization of the material, e.g.,through immobilization on a solid support, entrapment in a matrix (e.g.,a gel), lyophilization, or the like.

Kits also optionally include packaging materials or containers forholding a microfluidic device, system or reagent elements.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovemay be used in various combinations. All publications and patentdocuments cited in this application are incorporated herein by referencein their entirety for all purposes to the same extent as if eachindividual publication or patent document were specifically andindividually indicated to be incorporated by reference.

What is claimed is:
 1. A system for separating two or more analytes anddetermining a total analyte concentration or amount, the systemcomprising: (i) a microfluidic device comprising a body structure havinga plurality of microscale channels disposed therein, the microscalechannels comprising: (a) a first channel region; (b) a measurementchannel region fluidly coupled to the first channel region; (c) aseparation channel fluidly coupled to the first channel region; (d) afirst detection region, which first detection region is positionedproximal to the measurement channel region; and, (e) a second detectionregion, which second detection region is positioned proximal to theseparation channel; (ii) a fluid direction system fluidly coupled or aircoupled to the microfluidic device, which fluid direction systemdirects: (a) movement of at least two analytes through the first channelregion; (b) movement of the at least two analytes from the first channelregion into the measurement channel region; (c) movement of the at leasttwo analytes through the first detection region; (d) movement of the atleast two analytes from the first channel into the separation channel,thereby producing two or more separated analytes; and, (e) movement ofthe two or more separated analytes through the second detection region;and, (iii) a detection system positioned proximal to one or more of: thefirst detection region or the second detection region, which detectionsystem is configured to detect the at least two analytes in the firstdetection region, producing a total analyte signal, which total analytesignal corresponds to the total analyte concentration or amount, anddetects the two or more separated analytes in the second detectionregion, producing two or more analyte signals, which two or more analytesignals correspond to the two or more separated analytes.
 2. The systemfor separating two or more analytes of claim 1, wherein the systemfurther comprises a computer operably coupled to the detection system,which computer receives the total analyte signal and the two or moreanalyte signals and comprises software, which software comprises atleast a first instruction set and a second instruction set, which firstinstruction set determines the total analyte concentration or amountfrom the total analyte signal and which second instruction setdetermines the concentration or amount of each of the at least twoanalytes from the two or more analyte signals.
 3. The system of claim 2,wherein the computer comprises a third instruction set, which thirdinstruction set adds the two or more analyte signals, thus determiningthe total analyte concentration or amount.
 4. The system of claim 2,wherein the computer comprises a third instruction set, which thirdinstruction set determines a ratio or difference of the amount of atleast one of the at least two analytes to the total analyte amount or toa portion of the total analyte amount.
 5. The system of claim 1, whereinthe fluid direction system comprises one or more fluid control elementsfluidly coupled or air coupled to the plurality of microscale channels.6. The system of claim 5, wherein the one or more fluid control elementscomprise one or more of; a pressure source or an electrokineticcontroller.
 7. The system of claim 1, wherein the detection systemcomprises a single detector that monitors both the first detectionregion and the second detection region.
 8. The system of claim 1,wherein the detection system comprises a first detector positionedproximal to the first detection region and a second detector positionedproximal to the second detection region.
 9. A method of introducing arepresentative aliquot of a sample comprising at least two analytes intoa separation channel of a microfluidic device, the method comprisingflowing the sample through a first channel of a microfluidic device, thefirst channel being in fluid communication with the separation channelat an intersection, while flowing the sample through a detection region,monitoring a signal detected at the detection region, and injecting asample aliquot from the first channel into the separation channel whenthe signal indicates that all analytes in the sample have reached theintersection.
 10. The method of claim 9, wherein the sample aliquot isflowed through the first channel through the application of anelectrokinetic gradient.
 11. The method of claim 10, wherein the atleast two analytes in the sample aliquot have different electrokineticmobilities, wherein at least one of the at least two analytes comprisesa slowest analyte, and wherein all analytes have reached theintersection when the slowest analyte reaches the intersection.
 12. Themethod of claim 9, wherein subsequent to the sample aliquot beinginjected into the sample channel the analytes in the sample aliquot areelectrophoretically separated.
 13. The method of claim 9, wherein thedetection region is a subunit of the first channel.
 14. The method ofclaim 9, wherein the detection region is a subunit of a measurementchannel in fluid communication with the first channel.
 15. The method ofclaim 9, wherein the intersection comprises a portion of the separationchannel between two directly opposed portions of the first channel. 16.The method of claim 9, wherein the intersection comprises a portion ofthe separation channel between two offset portions of the first channel.17. The method of claim 9, wherein an assay performed in the firstchannel produces the sample.
 18. The method of claim 9, wherein flowingthe sample through the first channel comprises flowing sample aliquotfrom a first reservoir in fluid communication with the first channel toa second reservoir in fluid communication with the first channel. 19.The method of claim 9, wherein the signal comprises an optical signal.20. The method of claim 9, wherein the signal indicates that allanalytes in the sample aliquot have reached the intersection when thesignal reaches a constant value.
 21. The method of claim 9, wherein theinjection of a sample aliquot from the first channel into the separationchannel comprises a pinched injection.
 22. The method of claim 9,wherein the signal comprises a first signal that emanates from a firstmarker that flows slower than all analytes present in the samplealiquot, and detection of the first signal indicates that all analytesin the sample aliquot have reached the intersection.
 23. The method ofclaim 10, wherein the signal also comprises a second signal thatemanates from a second marker that flows faster than all analytespresent in the sample, and detection of the first and second signalsindicates that all analytes in the sample aliquot have reached theintersection.